Reduction of lipase activity in product formulations

ABSTRACT

The invention relates a method for producing a stable recombinant protein, comprising growing a non-naturally occurring host cell in a culture medium to produce a recombinant protein, and making a composition comprising the recombinant protein and a polysorbate. The production of endogenous lipoprotein lipase by the host cell is reduced. The endogenous lipoprotein lipase is present in the composition in a small amount, and is capable of degrading the polysorbate. The invention also relates to the relevant host cells and compositions, and preparation thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/105,925, filed Jun. 17, 2016, which is a U.S. national phaseapplication of International Application No. PCT/US2014/071234, filedDec. 18, 2014, claiming the benefit of U.S. Provisional Application No.61/917,555, filed Dec. 18, 2013, the contents of each of which areincorporated herein in their entireties for all purposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from the National Science Foundation(NSF) (Award No. CBET-0966644). The United States has certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates generally to making formulations of stablerecombinant proteins produced by non-naturally occurring host cells.

BACKGROUND OF THE INVENTION

Chinese hamster ovary (CHO) cells are integral to the $125 billionbiopharmaceutical market, which includes monoclonal antibodies (mAbs)and other therapeutic proteins. Recent sequencing of the Chinese hamsterand Chinese hamster ovary (CHO) cell genomes enables cell engineeringstrategies to address a wide variety of problems encountered inbiopharmaceutical manufacturing. One particular application involvesstudies of CHO host cell proteins (HCPs) that may be difficult to removefor a variety of reasons. The presence of HCPs is regulated for patientsafety concerns but may also have an impact on product quality in thecontext of formulation.

Polysorbates are a class of non-ionic surfactants that are added tobiopharmaceutical formulations to improve the stability of therapeuticproteins by limiting aggregation and surface adsorption. Monoclonalantibody formulations often incorporate a polysorbate such aspolysorbate 80 (PS-80) and polysorbate 20 (PS-20) to prolong theshelf-life of drug products. Polysorbate degradation over time canimpact the stability of those drug products.

There remains a need for improved mammalian host cells for producingstable recombinant proteins by, for example, mitigating polysorbatedegradation.

SUMMARY OF THE INVENTION

The present invention relates to host cells suitable for producing astable recombinant protein, compositions comprising the stablerecombinant proteins, methods for producing the stable recombinantproteins by the host cells, and methods for preparing the host cells andthe compositions.

According to a first aspect of the present invention, a non-naturallyoccurring host cell for producing a stable recombinant protein isprovided. The production of endogenous lipase, for example, lipoproteinlipase (LPL), by the host cell is reduced. Preferably, the lipase isLPL. The production of the endogenous lipase (e.g., LPL) may be reducedby at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%. No morethan about 1%, 5%, 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95% of therecombinant protein may be bound to the endogenous lipase (e.g., LPL).

The host cell may be a mammalian cell selected from the group consistingof CHO, 3T3, BHK, HeLa, NS0, HepG2, and derivatives thereof. Preferably,the host cell is a CHO cell.

The host cell may express an interfering RNA specific for the endogenouslipase (e.g., LPL). The interfering RNA may be selected from the groupconsisting of small interfering RNAs (siRNAs), short hairpin RNAs(shRNAs), and bifunctional RNAs. The interfering RNA may be encoded bythe genome of the host cell.

At least one copy of an endogenous gene encoding the endogenous lipase(e.g., LPL) may be knocked out from the genome of the host cell.Preferably, all copies of the endogenous gene are knocked out.

According to a second aspect of the invention, a composition isprovided. The composition comprises a stable recombinant protein and apolysorbate. The recombinant protein is produced by a non-naturallyoccurring host cell. The production of endogenous lipase (e.g., LPL) bythe host cell is reduced. The endogenous lipase (e.g., LPL) is presentin the composition in a small amount (e.g., less than about 10%, 5%, 1%,0.1%, 0.01%, 0.001% or 0.0001% by weight), and is capable of degradingthe polysorbate.

The composition may further comprise an inhibitor of the endogenouslipase (e.g., LPL). The inhibitor may inhibit the lipase (e.g., LPL)from binding the recombinant protein. The inhibitor may inhibit thelipase from degrading the polysorbate.

At least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95% of therecombinant protein in the composition may remain over a predeterminedperiod of time, for example, 1 day, 1 week, 2 weeks, 1 month, 3 months,6 months or 1 year.

Where the recombinant protein has a biological activity, at least about10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95% of the biological activityremains over a predetermined period of time, for example, 1 day, 1 week,2 weeks, 1 month, 3 months, 6 months or 1 year.

The recombinant protein may be an antibody, preferably a monoclonalantibody, more preferably a humanized antibody.

The polysorbate may comprise polysorbate 80, polysorbate 20, polysorbate40, polysorbate 60, polysorbate 65, or a combination thereof.

According to a third aspect of the invention, a method for producing astable recombinant protein is provided. The method comprises (a) growinga non-naturally occurring host cell in a culture medium to produce therecombinant protein, and (b) making a composition comprising therecombinant protein and a polysorbate. The production of endogenouslipase (e.g., LPL) by the host cell is reduced. The endogenous lipase(e.g., LPL) is present in the composition in a small amount (e.g., lessthan about 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or 0.0001% by weight), andis capable of degrading the polysorbate. The recombinant protein in thecomposition is stable.

According to the production method, the polysorbate may comprisepolysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 65, or a combination thereof. The production of theendogenous lipase (e.g., LPL) by the host cell may be reduced by atleast about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%.

According to the production method, at least about 95%, 90%, 80%, 70%,60%, 50%, 40%, 30%, 20% or 10% of the stable recombinant protein mayremain over a predetermined period of time, for example, 1 day, 1 week,2 weeks, 1 month, 3 months, 6 months or 1 year. Where the stablerecombinant protein is biologically active, at least about 95%, 90%,80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the biological activity mayremain over a predetermined period of time, for example, 1 day, 1 week,2 weeks, 1 month, 3 months, 6 months or 1 year. The recombinant proteinmay be an antibody, preferably a monoclonal antibody, more preferably ahumanized antibody.

According to the production method, the host cell may be a mammaliancell selected from the group consisting of CHO, 3T3, BHK, HeLa, NS0,HepG2, and derivatives thereof. Preferably, the host cell is a CHO cell.

The production method may further comprise expressing an interfering RNAspecific for the lipase (e.g., LPL) in the host cell. The interferingRNA may be selected from the group consisting of small interfering RNAs(siRNAs), short hairpin RNAs (shRNAs), and bifunctional RNAs. Theinterfering RNA may be encoded by the genome of the host cell.

The production method may further comprise knocking out at least onecopy of an endogenous gene encoding the endogenous lipase (e.g., LPL)from the genome of the host cell. Preferably, all copies of theendogenous gene encoding the endogenous lipase (e.g., LPL) are knockedout from the genome of the host cell.

The production method may further comprise removing the endogenouslipase (e.g., LPL) from the composition.

The production method may further comprise adding an inhibitor of theendogenous lipase (e.g., LPL) to the composition. The inhibitor mayinhibit the lipase (e.g., LPL) from binding the recombinant protein. Theinhibitor may inhibit the lipase from degrading the polysorbate.

According to a fourth aspect of the invention, a method for preparing anon-naturally occurring host cell suitable for producing a recombinantprotein is provided. The preparation method comprises reducing theproduction of endogenous lipase (e.g., LPL) by the host cell.

The preparation method may further comprise expressing an interferingRNA specific for the lipase (e.g., LPL) in the host cell. Theinterfering RNA may be selected from the group consisting of smallinterfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and bifunctionalRNAs. The interfering RNA may be encoded by the genome of the host cell.

The preparation method may further comprise knocking out at least onecopy, preferably all copies, of an endogenous gene encoding theendogenous lipase (e.g., LPL) from the genome of the host cell. The hostcell may be a mammalian cell selected from the group consisting of CHO,3T3, BHK, HeLa, NS0, HepG2, and derivatives thereof. Preferably, thehost cell is a CHO cell.

According to a fifth aspect of the invention, a method for preparing acomposition comprising a recombinant protein and a polysorbate isprovided. The preparation method comprises adding a polysorbate to aformulation comprising a recombinant protein produced by a non-naturallyoccurring host cell. The preparation method may further comprise addingan inhibitor of the endogenous lipase (e.g., LPL) to the composition.The production of endogenous lipase (e.g., LPL) by the host cell isreduced. The endogenous lipase (e.g., LPL) is present in the compositionin a small amount (e.g., less than about 10%, 5%, 1%, 0.1%, 0.01%,0.001% or 0.0001% by weight), and is capable of degrading thepolysorbate. The recombinant protein is stable in the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show (A) cell density, (B) cell viability, (C) extracellularHCP concentration, and (D) extracellular HCP productivity per cell over10 days for CHO cultures aged 136-500 days at the start of theexperiment. Error bars represent the standard error of the mean fromfour biological replicates of production culture sourced from a singlecryopreserved stock for each cell age. HCP productivity is unavailablefor cells aged 500 days at day 10 of the experiment, as no viable cellsremained in culture.

FIGS. 2A-B show heat map of extracellular CHO HCPs demonstratingstatistically significant varied expression by shotgun proteomicsrelative to expression in cells aged 136 days (yellow) with respect to(A) cell age and (B) CHO HCP productivity. Proteins exhibiting increasedexpression are shown in green, while those with decreased expression areshown in red. Color graduations are based on a logarithmic scale.Statistically indistinguishable pairwise comparisons denoted as^(a)equivalent between 136 and 251 days, and ^(b)equivalent between 19and 26 pg/cell (136 and 366 days).

FIGS. 3A-B show relative expression of (A) laminin subunit α-5 as anexample of cell age-dependent expression and (B) complement C3 as anexample of HCP productivity-dependent expression.

FIGS. 4A-D show representative 2DE images of extracellular CHO HCPscollected from day five of production culture from cells cultured for(A) 136, (B) 251, (C) 366, and (D) 500 days. Selected spots aremagnified to illustrate varied expression.

FIG. 5 shows a representative 2DE image of protein spots that exhibitedat least three-fold change in spot volume and were excised andidentified by MS. Only one spot is labeled for each protein. Molecularweight (MW) and isoelectric point (pI) labels approximated from thelocations of seven identified proteins.

FIGS. 6A-F show relative protein expression by 2DE and shotgunproteomics for six proteins with the lowest p-value by 2DE: (A)thrombospondin-1, (B) 78 kDa glucose-regulated protein, (C)nucleobindin-2, (D) basement membrane-specific heparan sulfateproteoglycan core protein, (E) lysosomal protective protein, and (F)cathepsin D. Only protein spots exhibiting at least a threefold changein relative spot volume are included in relative protein expression by2DE and multiple spots yielding the same protein identification werecombined for each image. Error bars represent the standard error of themean normalized spot volume from three biological replicates ofproduction culture sourced from a single cryopreserved stock for eachcell age. Statistical significance calculated by Tukey-Kramer HSD testwith respect to expression at cells cultured 136 days and denoted as **p<0.01 and * p<0.05 for both proteomic methods.

FIG. 7A-B show the impact of siRNA-mediated silencing on (A) LPLexpression and (B) total HCP expression. Error bars represent thestandard error of the mean from two biological replicates. LPLexpression was also measured in technical triplicate (n=6 totalmeasurements).

FIGS. 8A-B show cell culture performance with siRNA-mediated silencingof LPL. Measured cell culture attributes include (A) cell density and(B) cell viability. Error bars represent the standard error of the meanfrom two biological replicates.

FIG. 9 shows PS-80 digestion following incubation with extracellular CHOHCPs derived from control culture and following siRNA-mediated silencingof LPL. Control cultures digested an average of 0.2% of the initialPS-80 concentration prior to normalization. Error bars represent thestandard error of the mean from technical triplicate measurements.

FIGS. 10A-B show PS-80 digestion (A) depicts the amount of PS-80digested from a sample with no enzyme inhibitor and shows a high levelof PS-80 degradation compared to a sample where an inhibitor (Pefabloc)of LPL is added that significantly reduces the amount of PS-80 degraded(B) shows that the amount of PS-80 degraded by samples derived from CHOcells expressing an siRNA against LPL show a somewhat reduced amount ofPS-80 degradation compared to a cell only or a nonspecific controlsample.

FIG. 11 shows silver-stained reducing SDS-PAGE of non-induced (A) andinduced (B) LPL-producing E. coli cell cultures.

FIG. 12 shows Ni-NTA affinity purification of recombinant CHO LPL with250 mM imidazole step elution.

FIG. 13A-B show (A) the flow-through fraction A and elution fraction Bof LPL Ni-NTA affinity purification on silver-stained reducing SDS-PAGE,and (B) anti-His western blot of the LPL Ni-NTA affinity purificationflow-through fraction (A) and elution fraction (B).

FIG. 14 shows RP-HPLC gradient elution (45 min 0-100% acetonitrilelinear gradient, C₁₈ column, 1 mL/min) of refolded LPL (black) and LPLsolubilized in 6 M guanidine HCl (gray).

FIG. 15 shows representative chromatogram of ADAM-labeled degraded andnon-degraded polysorbate 80.

FIG. 16 shows digestion rate of polysorbate 80 by CHO LPL (produced inE. coli) in different solution conditions at 37° C. for 24 hours.

FIG. 17 shows digestion rate of polysorbate 20 by CHO LPL (produced inE. coli) in different solution conditions at 37° C. for 24 hours. Nomeasurable digestion was found at pH 6.0.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that lipoprotein lipase(LPL) is an endogenous CHO host cell protein (HCP) that co-purifies withdifferent monoclonal antibodies produced by CHO host cells and has anenzymatic activity that degrades polysorbate 80 (PS-80) and polysorbate20 (PS-20). In particular, the present invention relates to mitigatingexpression of endogenous lipase (e.g., LPL) by mammalian host cells(e.g., CHO cells) to improve stability of recombinant proteins producedby the host cells. There may be two general approaches to reducing oreliminating lipase (e.g., LPL) from a final drug product: reducing oreliminating lipase (e.g., LPL) that appears in an original host cellthrough a cell engineering approach, or adjusting purificationstrategies to specifically target lipase (e.g., LPL) removal. One mayalso add an inhibitor of the lipase (e.g., LPL) in a compositioncomprising a recombinant protein and a polysorbate.

The terms “protein” and “polypeptide” are used herein interchangeably,and refer to a polymer of amino acid residues with no limitation withrespect to the minimum length of the polymer. Preferably, the protein orpolypeptide has at least 20 amino acids. The definition includes bothfull-length proteins and fragments thereof, as well as modificationsthereof (e.g., glycosylation, phosphorylation, deletions, additions andsubstitutions).

The term “polynucleotide” used herein refers to a polymer of nucleotideresidues with no limitation with respect to the minimum length of thepolymer. Preferably, the polynucleotide has at least 60 nucleotides. Thepolynucleotide may be a DNA, cDNA or RNA molecule.

The term “variant” of a protein or polynucleotide used herein refers toa polypeptide having an amino acid or a polynucleotide having a nucleicacid sequence that is the same as the amino acid or nucleic acidsequence of the corresponding protein or polynucleotide except having atleast one amino acid or nucleic acid modified, for example, deleted,inserted, or replaced, respectively. A variant of a protein orpolynucleotide may have an amino acid or nucleic acid sequence at leastabout 80%, 90%, 95%, or 99%, preferably at least about 90%, morepreferably at least about 95%, identical to the amino acid sequence ornucleic acid of the protein or polynucleotide.

The term “lipase” used herein refers to a lipase gene family. Examplesinclude lipoprotein lipase (LPL), pancreatic lipase, hepatic lipase, andendothelial lipase.

Lipoprotein lipase (LPL) is a water soluble enzyme that hydrolyzestriglycerides in lipoproteins, such as those found in chylomicrons andvery low-density lipoproteins (VLDL), into two free fatty acids and onemonoacylglycerol molecule. It is also involved in promoting the cellularuptake of chylomicron remnants, cholesterol-rich lipoproteins, and freefatty acids.

The term “lipoprotein lipase (LPL)” used herein refers to a full lengthLPL protein, or a functional fragment or variant thereof. LPL proteinsequences and gene sequences in various species (e.g., human, mouse, ratand Chinese hamster) are known in the art. The actual or predicted LPLmRNA sequences of human, mouse, rat and Chinese hamster LPL can be foundin the GenBank database Accession Nos. NP_000228, NP_032535, NP_036730,and XP_007607328, respectively. A functional fragment or variant ofendogenous LPL produced by a host cell is capable of co-purifying with arecombinant protein, for example, a therapeutic protein, produced by thehost cell, and is capable of degrading a polysorbate.

The present invention provides a non-naturally occurring host cellsuitable for producing a recombinant protein. The production ofendogenous lipase by the host cell is reduced. The lipase is preferablylipoprotein lipase (LPL).

The recombinant protein may be bound to the endogenous lipase (e.g.,LPL). In some embodiments, no more than about 1%, 5%, 10%, 20%, 30%,40%, 60%, 70%, 80%, 90% or 95% the recombinant protein is bound to theendogenous lipase (e.g., LPL).

The term “production” used herein refers expression and secretion of aprotein by a host cell. The production of endogenous lipase (e.g., LPL)by a host cell may be reduced by at least about 10%, 20%, 30%, 40%, 60%,70%, 80%, 90% or 95%, preferably by at least about 20%, more preferablyby at least about 50%, most preferably by at least about 95%. In apreferred embodiment, the host cell produces no lipoprotein lipase.

The host cell may be a mammalian cell, preferably a mammalian cellsuitable for producing a recombinant protein. The host cell may beselected from the group consisting of 3T3, CHO, BHK, HeLa, HepG2 and NS0cells, and derivatives of these cells. Preferably, the host cell is aCHO cell. The host cell may be adherent or in suspension, preferably insuspension.

The production of the lipase (e.g., LPL) may be reduced by variousmethods known in the art. For example, the expression of the lipase(e.g., LPL) in host cells (e.g., CHO cells) may be reduced by using aninterfering RNA approach to reduce the amount of the lipase (e.g., LPL)transcript expression or by eliminating the lipase (e.g., LPL) gene fromthe genome of host cells (e.g., CHO cells) using a genome editingmethod.

The host cell may express an interfering RNA specific for the lipase(e.g., LPL). The interfering RNA is capable of interfering with theexpression of an endogenous gene encoding the lipase (e.g., LPL) andcausing reduced production of the lipase (e.g., LPL) by a host cellcomprising the interfering RNA when compared with that by a controlcell. The control cell is the same as the host cell except that itsendogenous lipase (e.g., LPL) production is not altered. The controlcell may be a naturally occurring cell. The interfering RNA may beselected from the group consisting of small interfering RNAs (siRNAs),short hairpin RNAs (shRNAs), and bifunctional RNAs.

Conventional RNA interference (RNAi) design and construction techniquesmay be used to make an interfering RNA specific for the lipase (e.g.,LPL) by targeting any segment of a lipase (e.g., LPL) mRNA. For example,an siRNA sequence may be complementary with a segment of a lipase (e.g.,LPL) mRNA sequence in a host cell. Where the lipase (e.g., LPL) mRNAsequence is not known in a host cell, a lipase (e.g., LPL) cDNA may beobtained from the host cell using conventional techniques known in theart. For example, a lipase (e.g., LPL) cDNA may be isolated from a hostcell and sequenced to define target regions for gene silencing based onpreviously published siRNA design guidelines. Various sequence segments,preferably conserved regions within the lipase (e.g., LPL) cDNA sequenceamong different species may be selected. For example, a LPL-specificsiRNA sequence may target an LPL mRNA segment sequence corresponding toan LPL gene sequence (XM_003499928.1) as set forth in Table 1. siRNAduplexes may be synthesized, and screened for silencing efficiency inhost cells, for example, CHO cells.

A lipase (e.g., LPL) specific interfering RNA may be introduced into ahost cell by various transfection methods. An effective lipase (e.g.,LPL) specific interfering RNA may be introduced in a host cell forstable expression using techniques known in the art, for example, viashRNA vectors. The host cell may express the lipase (e.g., LPL) specificinterfering RNA transiently or stably, preferably stably. The lipase(e.g., LPL) specific interfering RNA may be encoded by the genome of thehost cell.

The production of endogenous lipase (e.g., LPL) by a host cell may alsobe accomplished by knocking out at least one copy of an endogenous geneencoding the endogenous lipase (e.g., LPL) from the genome of the hostcell. In a preferred embodiment, all copies of the endogenous lipase(e.g., LPL) gene are knocked out from the genome of the host cell, andthe lipase (e.g., LPL) production is eliminated. Exemplary genomeediting methods include CRISPR/Cas9 and TALENs.

Successful reduction or elimination of endogenous lipase (e.g., LPL) maybe monitored using various techniques known in the art or customized forthis purpose. For example, production of endogenous lipase (e.g., LPL)by host cells may be reflected by degradation of PS-80 in a fatty acidassay using samples derived from the host cells. The degradationspecificity by the lipase (e.g., LPL) may be determined by using aninhibitor such as Pefabloc.

The host cell may further comprise a nucleic acid sequence encoding arecombinant protein. The nucleic acid sequence encoding a recombinantprotein may be integrated into the genome of the host cell. The hostcell may produce the recombinant protein, either transient or stably,preferably stably.

The present invention also provides a composition comprising a stablerecombinant protein and a polysorbate. The recombinant protein isproduced by the non-naturally occurring host cell of the presentinvention. The endogenous lipase (e.g., LPL) is present in thecomposition in a small amount (e.g., less than about 10%, 5%, 1%, 0.1%,0.01%, 0.001% or 0.0001%, preferably less than about 0.1%, by weight).The endogenous lipase (e.g., LPL) is capable of degrading thepolysorbate. Preferably, the composition comprises no lipase (e.g.,LPL).

The recombinant protein in the composition is stable. For example, atleast about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%, preferably atleast about 90%, more preferably at least about 95%, most preferably100%, of the recombinant protein remains over a predetermined period oftime, for example, about 1 day, 1 week, 2 weeks, 1 month, 3 months, 6months or 1 year, preferably about 3 months.

The recombinant protein may be a biopharmaceutical protein. It may be anantibody, preferably a monoclonal antibody, more preferably a humanizedantibody. Exemplary recombinant proteins include monoclonal antibodies(e.g., anti-EGFR mAb, anti-VEGF mAb, anti-Factor VIII mAb, anti-IgE mAb,anti-CD11a mAb, anti-interferon-β mAb, anti-TNFα mAb, anti-CD52mAb,anti-HER2mAb, and anti-CD20 mAb), human secreted alkaline phosphatase(SEAP), tissue plasminogen activator (tPA), α-glucosidase, laronidase,Ig-CTLA4 fusion, N-acetylgalactosamine-4-sulfatase, luteinizing hormone,erythropoietin, TNFα receptor fusion, Factor IX, follicle stimulatinghormone, β-glucocerebrosidase, and deoxyribonuclease I. The recombinantproteins may have various targets and mechanisms of action, for example,Alpha4/beta1/7 integrin, Alpha-galactosidase ERT, ATIII substitution,B-lymphocyte stimulator (BLyS), CD20, Complement C5 antagonist, EGF-R,EpCAM (cancer target) and CD3 (T cell recruitment), Epitope on RS virus,Factor VIII substitution, G-CSF receptor, Glycoprotein IIb/IIIaantagonist, Growth hormone (GH) receptor antagonist, hGH receptor, Humangluco-cerebrosidase ERT, Iduronate-2-sulfatase enzyme replacement, IL-1beta antagonist, IL-12 (p40) and IL-23, Insulin receptor, Insulin-likegrowth factor-1 (IGF-1) receptor agonist, Interferon alpha receptor,Interleukin-1 receptor (IL-1R) antagonist, Kallikrein, Keratinocytegrowth factor receptor, Neuromus-cular transmission (SNAP-25 cleavage),Substitution of coagulation Factor VIIa, Thrombopoietin (TPO) receptoragonist, TNF alpha antagonist, TNF-alpha (soluble and membrane bound),and Vascular endothelial growth factor (VEGF).

Where the recombinant protein has a biological activity, for example, atherapeutic effect, at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%,90% or 95%, preferably at least about 90%, more preferably at leastabout 95%, most preferably 100%, of the biological activity remains overa predetermined period of time, for example, about 1 day, 1 week, 2weeks, 1 month, 3 months, 6 months or 1 year, preferably about 3 months.

The polysorbate may be present at about 0.001-1%, preferably 0.01-0.02%% by weight in the composition of the present invention. The polysorbatemay be composed of one or more polyoxyethylene sorbitan monooleate fattyacid esters. For example, the polysorbate may comprise polysorbate 80,polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or acombination thereof.

In the composition, the recombinant protein may be bound to theendogenous lipase (e.g., LPL) produced by the same host cell. In someembodiments, no more than about 1%, 5%, 10%, 20%, 30%, 40%, 60%, 70%,80%, 90% or 95% of the recombinant protein in the composition is boundto the endogenous lipase (e.g., LPL).

The composition may further comprise an inhibitor of the endogenouslipase (e.g., LPL). The inhibitor may inhibit the lipase (e.g., LPL)from binding the recombinant protein. The inhibitor may inhibit thelipase from degrading the polysorbate.

The present invention also provides a method for producing a stablerecombinant protein by the non-naturally occurring host cell of thepresent invention. The method comprises growing the non-naturallyoccurring host cell in a culture medium to produce a recombinantprotein, and then making a composition comprising the recombinantprotein and a polysorbate. The production of an endogenous lipase (e.g.,LPL) by the host cell is reduced. The endogenous lipase (e.g., LPL) iscapable of degrading the polysorbate, and is present in the compositionin a small amount (e.g., less than about 10%, 5%, 1%, 0.1%, 0.01%,0.001% or 0.0001% by weight). Preferably, the lipase is LPL.

The recombinant protein in the resulting composition is stable. Forexample, at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%,preferably at least about 90%, more preferably at least about 95%, ofthe recombinant protein remains over a predetermined period of time, forexample, about 1 day, 1 week, 2 weeks, 1 month, 3 months, 6 months or 1year, preferably about 3 months.

According to the production method of the present invention, therecombinant protein may be a biopharmaceutical protein, for example, anantibody, preferably a monoclonal antibody, more preferably a humanizedantibody. The antibody may be in the form of monomers, oligomers orlarge aggregates, preferably monomers. Exemplary recombinant proteinsmay include monoclonal antibodies (e.g., anti-EGFR mAb, anti-VEGF mAb,anti-Factor VIII mAb, anti-IgE mAb, anti-CD11a mAb, anti-interferon-βmAb, anti-TNFα mAb, anti-CD52mAb, anti-HER2mAb, and anti-CD20 mAb),human secreted alkaline phosphatase (SEAP), tissue plasminogen activator(tPA), α-glucosidase, laronidase, Ig-CTLA4 fusion,N-acetylgalactosamine-4-sulfatase, luteinizing hormone, erythropoietin,TNFα receptor fusion, Factor IX, follicle stimulating hormone,β-glucocerebrosidase, and deoxyribonuclease I. The recombinant proteinsmay have various targets and mechanisms of action, for example,Alpha4/beta1/7 integrin, Alpha-galactosidase ERT, ATIII substitution,B-lymphocyte stimulator (BLyS), CD20, Complement C5 antagonist, EGF-R,EpCAM (cancer target) and CD3 (T cell recruitment), Epitope on RS virus,Factor VIII substitution, G-CSF receptor, Glycoprotein IIb/IIIaantagonist, Growth hormone (GH) receptor antagonist, hGH receptor, Humangluco-cerebrosidase ERT, Iduronate-2-sulfatase enzyme replacement, IL-1beta antagonist, IL-12 (p40) and IL-23, Insulin receptor, Insulin-likegrowth factor-1 (IGF-1) receptor agonist, Interferon alpha receptor,Interleukin-1 receptor (IL-1R) antagonist, Kallikrein, Keratinocytegrowth factor receptor, Neuromus-cular transmission (SNAP-25 cleavage),Substitution of coagulation Factor VIIa, Thrombopoietin (TPO) receptoragonist, TNF alpha antagonist, TNF-alpha (soluble and membrane bound),and Vascular endothelial growth factor (VEGF). Where the recombinantprotein has a biological activity (e.g., a therapeutic effect), at leastabout 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%, preferably at leastabout 90%, more preferably at least about 95%, of the biologicalactivity remains over a prescribed period of time, for example, about 1day, 1 week, 2 weeks, 1 month, 3 months, 6 months or 1 year, preferablyabout 3 months.

According to the production method of the present invention, the hostcell produces at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or95%, preferably at least about 20%, more preferably at least about 50%,less lipase (e.g., LPL) than a control cell. The control cell is thesame as the host cell except that its endogenous lipase (e.g., LPL)production is not altered. The control cell may be a naturally occurringcell. Preferably, the host cell does not produce the lipase (e.g., LPL).

The production method may further comprise reducing the production ofthe endogenous lipase (e.g., LPL) by the host cell. For example, theproduction method may further comprise expressing an interfering RNA inthe host cell or knocking out at least one copy, preferably all copies,of an endogenous gene encoding the endogenous lipase (e.g., LPL) fromthe genome of the host cell. The interfering RNA may be selected fromthe group consisting of small interfering RNAs (siRNAs), short hairpinRNAs (shRNAs), and bifunctional RNAs.

Where the host cell produces endogenous lipase (e.g., LPL), the producedendogenous lipase (e.g., LPL) may be bound to the recombinant protein.The endogenous lipase (e.g., LPL) bound to the recombinant protein maybe removed from the recombinant protein by techniques known in the art.For example, the bound lipase (e.g., LPL) may be removed from therecombinant protein by washing, adding an excipient, or using anaffinity column. The production method may further comprise removing theendogenous lipase (e.g., LPL) bound to the recombinant protein, eitherbefore or after making the composition comprising the recombinantprotein and the polysorbate. In some embodiments, the percentage of therecombinant protein in the composition that is bound to the endogenouslipase (e.g., LPL) may be reduced by at least about 10%, 20%, 30%, 40%,60%, 70%, 80%, 90% or 95%, preferably by at least about 20%, morepreferably by at least about 50%, most preferably by at least about 95%.In a preferred embodiment, the recombinant protein in the composition isnot bound to the endogenous lipase (e.g., LPL).

Where the composition comprises the endogenous lipase (e.g., LPL), theproduction method may further comprise removing the endogenous lipase(e.g., LPL) from the composition. The lipase (e.g., LPL) removal may beaccomplished by techniques known in the art. For example, the endogenouslipase (e.g., LPL) may be removed from the composition by adding anexcipient or using an affinity column. In some embodiments, thepercentage of the endogenous lipase (e.g., LPL) in the composition isreduced by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%,preferably by at least about 20%, more preferably by at least about 50%,most preferably by at least about 95%. In a preferred embodiment, theresulting composition comprises no endogenous lipase (e.g., LPL).

The production method may further comprise adding an inhibitor of theendogenous lipase (e.g., LPL) to the composition. The inhibitor mayinhibit the lipase (e.g., LPL) from binding the recombinant protein. Theinhibitor may inhibit the lipase from degrading the polysorbate.

For each non-naturally occurring host cell suitable for producing arecombinant protein according to the present invention, a method forpreparing the host cell is provided. The preparation method comprisesreducing the production of endogenous lipase (e.g., LPL) by the hostcell. Preferably, the lipase is LPL. The production of the endogenouslipase (e.g., LPL) may be reduced by at least about 10%, 20%, 30%, 40%,60%, 70%, 80%, 90% or 95%, preferably by at least about 20%, morepreferably by at least about 50%. In a preferred embodiment, the hostcell does not produce the endogenous lipase (e.g., LPL). The host cellmay be a mammalian cell selected from the group consisting of CHO, 3T3,BHK, HeLa, NS0, HepG2, and derivatives thereof. Preferably, the hostcell is a CHO cell. The preparation method may further compriseexpressing an interfering RNA specific for the lipase (e.g., LPL) in thehost cell. The interfering RNA may be a small interfering RNA (siRNA),short hairpin RNA (shRNA), or bifunctional RNA. The interfering RNA maybe encoded by the genome of the host cell. Alternatively, thepreparation method may further comprise knocking out at least one copy,preferably all copies, of an endogenous gene encoding the endogenouslipase (e.g., LPL) from the genome of the host cell.

For each composition according to the present invention, a method forpreparing the composition is provided. The preparation method comprisesadding a polysorbate to a formulation comprising a recombinant proteinproduced by the non-naturally occurring host cell of the presentinvention. Preferably, the lipase is LPL. The production of endogenouslipase (e.g., LPL) by the host cell is reduced by, for example, at leastabout 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%, preferably by atleast about 20%, more preferably by at least about 50%. The endogenouslipase (e.g., LPL) is present in the composition in a small amount(e.g., less than about 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or 0.0001% byweight), and is capable of degrading the polysorbate. The recombinantprotein is stable in the composition. For example, at least about 10%,20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%, preferably at least about 90%,more preferably at least about 95%, of the recombinant protein remainsover a predetermined period of time, for example, about 1 day, 1 week, 2weeks, 1 month, 3 months, 6 months or 1 year, preferably about 3 months.The recombinant protein may be an antibody, preferably a monoclonalantibody, more preferably a humanized antibody. Where the recombinantprotein has a biological activity (e.g., a therapeutic effect), at leastabout 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 95%, preferably at leastabout 90%, more preferably at least about 95%, of the biologicalactivity remains over a predetermined period of time, for example, about1 day, 1 week, 2 weeks, 1 month, 3 months, 6 months or 1 year,preferably about 3 months. The polysorbate may be composed of one ormore polyoxyethylene sorbitan monooleate fatty acid esters. For example,the polysorbate may comprise polysorbate 80, polysorbate 20, polysorbate40, polysorbate 60, polysorbate 65, or a combination thereof. Theproduction method may further comprise adding an inhibitor of theendogenous lipase (e.g., LPL) to the composition. The inhibitor mayinhibit the lipase (e.g., LPL) from binding the recombinant protein. Theinhibitor may inhibit the lipase from degrading the polysorbate.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a percentage, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate.

EXAMPLE 1 Expression of Difficult-to-Remove Host Cell Protein ImpuritiesDuring Extended Chinese Hamster Ovary Cell Culture and their Impact onContinuous Bioprocessing

During biopharmaceutical manufacturing, Chinese hamster ovary (CHO)cells produce hundreds of extracellular host cell protein (HCP)impurities, which must be removed from the therapeutic product bydownstream purification operations to ensure patient safety. A sub-setof 118 of these HCPs have been reported as exceptionally difficult toremove during downstream purification because they co-purify due toretention characteristics on chromatographic media and/orproduct-association through strongly attractive interactions to thetherapeutic protein. As the biopharmaceutical industry moves towardscontinuous bioprocessing, it is important to consider the impact ofextended culture of CHO cells on the expression of extracellular HCPimpurities, especially those HCPs known to challenge downstreampurification. Two complementary proteomic techniques, two-dimensionalelectrophoresis (2DE) and shotgun, were applied to detect variations inthe extracellular CHO HCP profile over 500 days of culture. In total, 92HCPs exhibited up to 48-fold changes in expression, with 34 of theseHCPs previously reported as difficult to purify. Each proteomictechnique detected differential expression by a distinct set of HCPs,with 10 proteins exhibiting significant variable expression by bothmethods. This study presents the impact of cell age on the extracellularCHO HCP impurity profile and identifies HCPs with variable expressionlevels, which warrant further investigation to facilitate theirclearance in downstream purification.

1. Introduction

Typically, therapeutic proteins are secreted into the extracellularmedia along with hundreds of endogenous host cell protein (HCP)impurities, comprising both secreted proteins and intracellular proteinsreleased during cell death. Purification processes clear theseextracellular CHO HCPs from the therapeutic proteins because even lowlevels of HCP impurities have the potential to cause adverse patientreactions. A subset of these HCPs are difficult to remove duringdownstream purification because they exhibit product association withmAbs or similar retention to mAbs on chromatographic media. Variableexpression during upstream cell culture, which changes the compositionof the impurity profile fed into downstream purification, can increasethe complexity of removing these difficult-to remove impurities becausethe composition of HCPs generated upstream has been shown to impactimpurity clearance during downstream purification. HCPs with variableexpression during cell culture in addition to co-purifying acrosspurification are particularly likely to persist across purificationoperations into the final drug product. Consequently, it is necessary toidentify specific HCPs that are likely to vary during cell culture andto characterize how these specific impurities are cleared in downstreamoperations.

Analysis of extracellular CHO HCPs can be achieved by proteomictechniques including two-dimensional electrophoresis (2DE) and shotgunmethods, which are complementary techniques that can be applied toseparate and quantify proteins and peptides, respectively. Proteomictechniques have been applied to track HCP clearance across variouspurification operations and to identify specific HCPs that are difficultto remove from therapeutic products during downstream purification. Forexample, HCPs likely to persist across capture by Protein Achromatography have been demonstrated by 2DE and shotgun methods, whileHCPs likely to co-purify across alternative resin moieties have beenidentified by shotgun techniques. 2DE methods have also been applied toidentify. HCPs likely to evade clearance due to strongly attractiveinteractions with mAbs. In total, 118 HCP impurities have been reportedas exceptionally difficult to purify during downstream purification inprevious work. Variable expression of these difficult-to-remove HCPsduring upstream operations may further challenge their clearance indownstream purification.

In the context of upstream biopharmaceutical manufacturing, inputfactors such as temperature and media composition have been shown toexhibit a limited effect on the extracellular CHO HCP impurity profile;however, extracellular HCP expression is significantly impacted byconditions that decrease cell viability. As manufacturing platformsevolve towards continuous bioprocessing, it is important to evaluate theimpact of additional upstream factors on the HCP composition,particularly with regard to HCPs that are difficult to remove duringdownstream purification. Continuous bioprocessing represents aninnovative technology characterized by integrating perfusion cellculture with continuous chromatography and other unit operations, andoffers the potential for decreased cost and increased flexibilitycompared to traditional manufacturing platforms. Perfusion cultures havebeen demonstrated for over 60 days of continuous operation, during whichthe HCP profile may change through genetic modifications and phenotypicchanges. Beckmann et al. applied 2DE to study the intracellular CHOproteome over 420 days of culture and demonstrated variable expressionof several intracellular HCPs, including increased expression of severalglycolytic enzymes and anti-stress proteins. Consequently, thecomposition of extracellular CHO HCP impurities requiring removal from atherapeutic product may evolve over extended cell culture duringcontinuous bioprocessing and challenge removal during downstreamoperation. If purification operations are not designed to remove thefull range of HCP levels resulting from such variable expression,product quality could be negatively impacted. Variable expression ofHCPs that have been previously demonstrated as difficult to removeduring downstream purification poses additional level of complexity forimpurity clearance.

This study is the first to report changes in the extracellular CHO HCPcomposition associated with cell age upstream and to identify HCPs withvariable expression that may impact impurity clearance during downstreamprocesses, with a particular focus on HCPs that have been previouslyreported as difficult to remove during downstream purification. Cellscultured for four different durations of up to 500 days were compared by2DE and shotgun proteomics. In total, 630 unique proteins wereidentified by the two techniques, with 92 extracellular CHO HCPsdemonstrating variable expression relative to the shortest cultureduration (136 days), Additionally, 37% of HCPs exhibiting variedexpression in this work have previously been identified as potentiallydifficult to remove by downstream processes. These proteins represent asub-set of the extracellular CHO proteome, which may be especiallydifficult to remove; further investigation of these HCPs couldfacilitate improved clearance in downstream purification.

2. Materials and Methods

2.1 Extended Culture of CHO Cells

A null CHO-K1 cell line (ATCC, Manassas, Va.) was adapted to serum-free,suspension culture in 125 mL shake flasks containing 20-30 mL SFM4CHOmedium (Hyclone Laboratories Inc., Logan, Utah). The adaptation processoccurred over 136 days, after which the cells were subjected to extendedculture with routine passaging at 3-5 day intervals in a 37° C. cellculture incubator with 5% CO₂ and 80% relative humidity. At fourtime-points during culture (136, 251, 366, and 500 days), a portion ofcells was removed and cryopreserved at 0.5-2.3×10⁶ cells/mL in 7.5%dimethyl sulfoxide (Sigma-Aldrich Chemical Co., St. Louis, Mo.), 50%conditioned media, and 42.5% fresh media. These cryopreserved cellstocks from four different cell ages were stored using polypropylenecryogenic vials (Corning Inc., Corning, N.Y.) in liquid nitrogen untilfurther use.

2.2 CHO Cell Production Cultures

The four cryopreserved cell stocks were thawed in parallel, transferredto 125 mL shake flasks containing 20 mL media, and cultured for 11 daysuntil typical growth rates were regained. Cultures from each cell agewere then seeded at 5×10⁴ cells/mL and incubated with orbital agitationfor 10 days in a 37° C. cell culture incubator with 5% CO₂ and 80%relative humidity. Cells were counted daily using a Fuchs Rosenthalhemocytometer (Hausser Scientific Co., Horsham, Pa.) with viabilitydetermined by the Trypan blue exclusion method. Portions of theextracellular CHO HCPs were harvested daily and separated from theresidual cells by centrifugation (180 g, 10 min) and stored at −20° C.until further use. All samples were analyzed for total proteinconcentration by Bradford assay (Pierce Chemical, Rockford, Ill.). Fourbiological replicates of production culture sourced from a singlecryopreserved stock were performed for each cell age Production culturereplicates were performed from two separate experiments, each with anindependent thaw of the cryopreserved stocks.

2.3 Quantitative Shotgun Proteomics

Samples containing 200 μg of extracellular CHO HCP harvested on day fiveof the production culture were precipitated with methanol by previouslyoptimized methods (Valente et al., 2014, Biotechnol. J. 9:87-99) andresolubilized in 100 mM triethylammonium bicarbonate buffer(Sigma-Aldrich Chemical Co.). Residual detergent was removed byDetergentOUT™ GBS10-800 detergent removal kit (G-Biosciences, St. Louis,Mo.) according to the manufacturer's protocol. Triethylammoniumbicarbonate buffer was removed by drying protein pellets in a SpeedVac™vacuum concentrator (Thermo Fisher Scientific Inc., Waltham, Mass.) andprotein pellets were resolubilized in dissolution buffer with denaturant(both from iTRAQ™ reagent kit, AB Sciex, Framingham, Mass.). For eachsample, 80 μg protein was reduced, alkylated, digested and labeledaccording to the manufacturer's protocol. HCPs from cells cultured for136, 251, 366, and 500 days were labeled with iTRAQ™ tags (isobariclabels to quantify relative expression) 117, 116, 115, and 114,respectively. Protein concentration was measured by Bradford assay(Thermo Fisher Scientific Inc., Rockford, Ill.) both from the cellculture supernatant and following detergent removal.

Peptide separation by reversed phase high performance liquidchromatography (RPHPLC) was performed as described previously (Valenteet al., 2014, Biotechnol. J. 9:87-99). Briefly, peptides were firstseparated by high-pH RP-HPLC on an Agilent 1100 (Agilent Technologies,Santa Clara, Calif.) using a 0.5 mL Varian PLRP-S column (AgilentTechnologies). Elution was achieved by an acetonitrile gradient in 50 mMammonium hydroxide (Avantor, Center Valley, Pa.), with eluate pooledinto 15 fractions, which were further separated by low-pH RP-HPLC usinga Tempo LCMALDI spotter (Eksigent, Dublin, Ireland) with a 1.2 μL CapRodRP-18E capillary column (Merck KGaA, Darmstadt, Germany). Peptides wereeluted by an acetonitrile gradient in 0.1% trifluoroacetic acid(Avantor) and eluate was spotted onto target plates withα-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Chemical Co.) matrix.

Data were collected by matrix-assisted laser desorption/ionizationtandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) asdescribed previously (Valente et al., 2014, Biotechnol. J. 9:87-99) onan AB Sciex 5800 MALDI-TOF/TOF Analyzer. Data were acquired in positiveion MS reflector mode and MS/MS with a maximum of 8 precursors per spot,and then submitted for database searches through ProteinPilot softwarev3.0 (AB Sciex). Spectra were searched against translations of the CHOgenome (Xu et al., 2011, Nat. Biotechnol. 29:735-741) with cysteinealkylation by methyl methanethiosulfonate. Peptide identifications with95% confidence or greater and protein identifications containing atleast one significant (p≤0.05) unique peptide were accepted.

Relative protein quantitation and statistical analysis were performedthrough ProteinPilot software with automatic bias and backgroundcorrection. Only peptides that were distinct to each protein wereconsidered for relative quantitation. Proteins were defined as variablyexpressed if (1) they included at least four significant (p≤0.05)peptides, (2) they exhibited statistically significant differentialexpression (p≤0.01) at any cell age relative to expression in cellscultured for 136 days, and (3) they satisfied a 5% false discovery ratecriterion (q≤0.05).

2.4 2DE Proteomics

2DE was performed as described previously (Valente et al., 2012,Electrophoresis 33:1947-1957) using 200 or 300 μg extracellular CHO HCPfrom each cell age with HCPs harvested on day five of the productionculture and protein concentration measured from the cell culturesupernatant by Bradford assay. HCPs were precipitated by tricholoraceticacid (Fisher Scientific, Fair Lawn, N.J.) according to previouslyoptimized methods (Valente et al., 2014, Biotechnol J 9:87-99). Briefly,proteins were precipitated with 15% tricholoracetic acid, washed withacetone, and resolubilized in rehydration solution comprising 8 mMtris(hydroxymethyl)aminomethane (Bio-Rad Laboratories, Hercules,Calif.), 8 M urea (Bio-Rad Laboratories), 30 mM dithiothreitol (Bio-RadLaboratories), 2%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Sigma-AldrichChemical Co.), 0.4% BioLytes (Bio-Rad Laboratories), and tracebromophenol blue (Bio-Rad Laboratories). Resolubilized proteins wereused to rehydrate 18 cm, pH 3-10 nonlinear Immobiline DryStrips (GEHealthcare, Chalfont St. Giles, United Kingdom) and isoelectric focusing(IEF) was performed using a PROTEAN IEF Cell (Bio-Rad Laboratories) for100,000 Vh, after which IEF gels were sequentially equilibrated withdithiothreitol and iodoacetamide (Sigma-Aldrich Chemical Co.). SDS-PAGEwas performed using 13% T, 2.6% C polyacrylamide slab gels, which werestained with SYPRO Ruby (Molecular Probes, Eugene, Oreg.) and imaged onan FLA-3000 Fluorescent Image Analyzer (Fujifilm Corp., Tokyo, Japan).Gel images were analyzed and compared using ImageMaster 2D PlatinumSoftware v5.0 (GE Healthcare). Spots were detected using the auto-detectfeature and manually edited to remove artifacts, while spot matching wasperformed manually by comparing images. The relative spot volume wascalculated by normalizing the volume of each protein spot to the totalspot volume detected in each image. Spots exhibiting at least athree-fold change in relative volume across the four cell ages wereexcised for identification.

Excised spots were analyzed by MALDI-TOF/TOF MS as described previously(Valente et al., 2014, Biotechnol. J. 9:87-99) on an AB Sciex 4800MALDI-TOF/TOF Analyzer. Data were acquired in positive ion MS reflectormode and MS/MS, and then submitted for Mascot v2.2 (Matrix Science Ltd.,London, UK) database searches through GPS Explorer software v3.6 (ABSciex). Spectra were searched against translations of the CHO genome andthe NCBInr database with 50 ppm mass tolerance, and oxidation ofmethionines and carbamidomethylation of cysteines allowed as variablemodifications. Identifications with 95% confidence or greater wereaccepted.

2DE analysis was performed on three biological replicates of productionculture sourced from a single cryopreserved stock for each cell age. Therelative spot volumes from spots with varied expression were tested forstatistical significance by ANOVA using JMP Pro 10 (SAS Institute Inc.,Cary, N.C.). Only protein spots that were detected on all threereplicates of each cell age were considered, and multiple spots yieldingidentification of a single protein were collated prior to statisticalanalysis. Proteins identified were defined as variably expressed if theyexhibited statistically significant differential expression (p≤0.1). Forproteins with variable expression, relative protein expression wasdetermined by normalizing the relative spot volume of each spot to thecorresponding relative spot volume at day 136. The statisticalsignificance of pairwise comparisons to cells cultured for 136 days wascalculated by the Tukey-Kramer HSD test using JMP Pro 10.

3. Results

3.1 Cell Growth with Varied Cell Age

To streamline proteomic analysis without interference from anoverexpressed product, CHO-K1 cells were used because null CHO cellshave demonstrated equivalent HCP compositions to recombinant proteinproducing cell lines (Grzeskowiak et al., 2009, Protein Expr. Purif.66:58-65; Jin et al., 2009, Biotechnol. Bioeng. 105:306-316; Tait etal., 2011, Biotechnol. Bioeng. 109:971-982). Cryopreserved stocks ofCHO-K1 cells aged 136, 251, 366, and 500 days were cultured for 10 days,with daily analysis of viable cell density (FIG. 1A), cell viability(FIG. 1B), extracellular CHO HCP concentration (FIG. 1C), andextracellular CHO HCP productivity per cell (FIG. 1D). As these datawere collected from null CHO cells, the HCP productivity per cellrepresented in FIG. 1D refers to the concentration of extracellular CHOHCPs normalized to the viable cell density over 10 days of productionculture, and not the productivity of a recombinant product. Cellscultured for 136, 251, and 500 days all exhibit typical exponentialgrowth over the first five days of culture and achieve a maximum celldensity at day seven (FIG. 1A), followed by a decrease in cell densitycorresponding to a loss of cell viability (FIG. 1B). For these threecell ages, the growth rate and maximum cell density correlate withculture age, with older cells demonstrating increased growth.Conversely, cells cultured for 366 days show a different growth profile,attaining a maximum cell density after four days in culture, followed bya steady decrease in cell density over the remainder of the productionculture (FIG. 1A). Statistically equivalent viability is observed acrossall cell ages for the first five days of culture, after which viabilitydecreases with increasing cell age (FIG. 1B). Cells cultured for 366days exhibit the greatest CHO HCP concentration (FIG. 1C), resulting inthe greatest CHO HCP productivity (FIG. 1D).

3.2 Shotgun Proteomics

On day five of the production culture, supernatant from all four cellages was collected for iTRAQ™ shotgun proteomics. In total, 3658significant (p≤0.05) peptides were detected, resulting in identificationof 630 unique HCPs, of which 85 HCPs (13%) demonstrated variedexpression (p≤0.01, with q≤0.05) relative to expression in cellscultured for 136 days. Variable expression was observed in 47, 59, and50 HCPs from cells cultured for 251, 366, and 500 days, respectively.65% of HCPs with varied expression exhibited at least a three-foldchange in expression level, with increases of up to 44-fold (atrialnatriuretic factor) and decreases of up to 48-fold (complement C3)observed.

Of the 85 HCPs that exhibited variable expression by iTRAQ™ shotgunproteomics, 24 demonstrated expression that correlated with cell age(FIG. 2A). For example, laminin subunit beta-1 expression increased withcell age, while expression of chondroitin sulfate proteoglycan 4decreased with cell age. Eight of the 24 proteins with cellage-dependent expression, such as lysosomal protective protein,exhibited a statistically significant decrease in protein expression forcell ages spanning 136 to 366 days, followed by a slight, significantincrease in protein expression between 366 and 500 days (FIG. 2A, bottompanel). Additionally, 20 proteins showed expression that correlated withextracellular CHO HCP productivity (FIG. 2B), such as complement C3,which exhibits a positive correlation and 78 kDa glucose-regulatedprotein, which demonstrates a negative correlation. FIG. 3 shows therelative protein expression of laminin subunit α-5 (FIG. 3A) andcomplement C3 (FIG. 3A) as examples of cell age-dependent expression andproductivity-dependent expression, respectively. The remaining 41proteins with varied expression either demonstrated variable expressionthat did not correlate with cell age or productivity, exhibited maximaor minima in cells aged 251 or 500 days, or lacked enough statisticallysignificant data to elucidate a correlation.

3.3 2DE Proteomics

Representative 2DE images of extracellular CHO HCPs harvested on dayfive of production culture are shown, with magnified images of selectspots to illustrate variable expression, across cells cultured for 136(FIG. 4A), 251 (FIG. 4B), 366 (FIG. 4C), and 500 (FIG. 4D) days. Acrossthree biological replicates of production culture sourced from a singlecryopreserved stock for each cell age, 50 protein spots exhibited atleast a three-fold change in spot volume and were subsequentlyidentified by MS. These 50 spots resulted in the identification of 32unique proteins (FIG. 5, with identifications listed in Table 2), ofwhich 17 demonstrated statistically significant variations in expression(p<0.1) by ANOVA, including seven HCPs that did not exhibit variableexpression by shotgun proteomics (Table 2). The significance criterionapplied to 2DE (p<0.1) was less stringent than that for shotgun (p<0.01)because the decreased number of ANOVA required for 2DE analysis (32proteins compared to 631 for shotgun) reduces the absolute number offalse positive identifications at a given confidence level. Relative HCPexpression agrees between 2DE and shotgun methods for both the sixproteins with the lowest p-values by 2DE (FIG. 6) and the remainingproteins with significant varied expression by 2DE. 2DE exhibitsdecreased magnitudes of change in relative HCP expression compared toshotgun proteomics, with a maximum increase of 6-fold exhibited bythrombospondin-1 and a maximum decrease of 9-fold demonstrated bylysosomal protective protein.

3.4 Difficult-to-Remove HCPs

From cells cultured for 251, 366, and 500 days, 92 unique HCPs exhibitedvariable expression relative to expression in cells cultured for 136days by either shotgun (85 proteins) or 2DE (17 proteins) methods, with10 proteins showing varied expression by both techniques. Of these 92HCPs, 34 have previously been reported as potential purificationchallenges (Table 3). Seventeen of these HCPs are difficult to removebecause they were shown to exhibit strongly attractive interactions withmAbs under Protein A solution conditions (Levy et al., 2014, Biotechnol.Bioeng. 111:904-912), while 15 of these HCPs were previously detected inProtein A eluate (Doneanu et al., 2012, MAbs 4:24-44; Hogwood et al.,2013, Biotechnol. Bioeng. 110:240-251). The remainingdifficult-to-remove HCPs demonstrated similar retention characteristicsto therapeutic products on a variety of polishing resins, includingmixedmode, cation exchange, and multimodal chromatography (MMC) ligands(Joucla et al., 2013, J Chromatogr. B 942-943:126-133; Pezzini et al.,2011, J. Chromatogr. A 1218:8197-8208).

4. Discussion

The specific growth rate and maximum viable cell density increased withcell age for cells cultured for 136, 251, and 500 days, consistent withprevious research. Conversely, cells cultured for 366 days achieved thelowest viable cell density and greatest extracellular CHO HCPconcentration. Cells cultured for 366 days also exhibited the greatestHCP productivity, which can be attributed to enhanced HCP productionand/or secretion rather than release of intracellular HCPs by celllysis, because cells cultured for 366 and 500 days exhibit equivalentviability throughout the production culture. The unique cell growthdemonstrated by cells cultured for 366 days is unexpected as cultureconditions were consistent throughout the 500 days of culture and cellswere not subjected to additional external stress at 366 days.

Because CHO cells are highly amenable to genetic modifications, thedecreased cell growth exhibited by cells cultured for 366 days mayresult from random mutations resulting in unfavorable genomic changesbetween 251 and 366 days of culture. This hypothesis is supported byprevious works that have documented the loss of specific productivity ofa therapeutic product due to genetic instability, even in populationsoriginating from a single CHO cell. Furthermore, it is reasonable thatadditional genetic mutations may have occurred between 366 and 500 daysthat either reversed the unfavorable mutations or induced additionalmutations resulting in a phenotype with recovered cell density. As theseresults were determined from four biological replicates of productionculture sourced from a single cryopreserved stock for each cell age, thereproducibility of CHO cell adaptation and extended cell culture remainsunknown. Given the unique growth demonstrated by cells cultured for 366days, it would be useful to replicate the entire process of CHO celladaptation and extended culture to evaluate the reproducibility of theobserved decrease in cell growth at 366 days of culture.

In total, 92 HCPs exhibited varied expression, with 92% detected byiTRAQ™ shotgun proteomics, 18% detected by 2DE, and 11% detected by bothtechniques. This finding is in agreement with previous work using thesame complementary proteomic methods. For example, comparisons ofHCT-116 cell lysates with varied p53 expression, Escherichia colilysates with varied induction levels and times, and human lung squamouscarcinoma versus normal tissue showed 85-97% of differentially expressedproteins were identified by iTRAQ™, 13-35% were identified by 2DE, and4-29% were identified by both methods. Increased detection by shotgunproteomics compared to 2DE is reasonable because shotgun proteomicsidentifies peptides, while identification of proteins by 2DE is limitedby reduced throughput and visual detection of spots during manualexcision. For the 10 proteins identified by both methods in this work,relative expression trends were in agreement across techniques, withiTRAQ™ shotgun proteomics exhibiting an increased magnitude ofexpression change compared to 2DE, which is consistent with previousreports.

Because 2DE detects predominantly proteins and shotgun workflows detectpeptides, identification of distinct groups of proteins by eachtechnique is expected because the varied physicochemical properties ofeach protein result in different resolution between the two differentmethods. For example, 2DE is limited in detecting proteins with extremehydrophobicity, molecular weight, or isoelectric point. Consequently,the majority (57%) of the seven proteins that exhibited variedexpression by 2DE alone (Table 2) are cytoplasmic, while less than 7% ofproteins identified as differentially expressed by shotgun proteomicsare classified as cytoplasmic. Shotgun methods begin with a proteolyticdigestion of proteins into peptides and are therefore limited in theirability to measure protein-level changes because a single peptide mayoriginate from multiple proteins or protein isoforms, while multiplepeptides derived from the same protein may show varied quantificationdue to the varied physicochemical properties of each peptide.

Differences between the precipitation methods required to maximizeproteome coverage for each technique may also have contributed to theunique set of proteins detected by each method. For example, three ofthe HCPs only identified by 2DE (cofilin-1, glutathione transferaseclass pi, nucleoside diphosphate kinase B) are relatively small withmolecular weight less than 25 kDa. One limitation of organic solventprecipitation, such as the methanol precipitation used to prepare HCPsfor shotgun proteomics in this work, is decreased efficiency of recoveryof small proteins, while the TCA precipitation used to prepare HCPs for2DE is less dependent on protein size. Consequently, the seven HCPs thatwere not detected by shotgun proteomics may have exhibited betterrecovery during sample preparation for 2DE compared to shotgun analysis.

Three of the extracellular CHO HCPs identified in this study (cofilin-1,glutathione transferase, and peroxiredoxin-1) had previously beenidentified as being variably expressed within the intracellular proteomeas a result of extended culture. As the present study examined theextracellular proteome from high-viability (>95%) cultures, it isexpected that few proteins would overlap with those identified byBeckmann et al. given the substantial difference in HCP compositionbetween intracellular and extracellular proteomes. The limited overlapbetween the two studies supports the hypothesis that the majority ofHCPs identified in this work exhibit variable expression due to changesin expression or secretion rather than release of intracellular proteinsby cell lysis.

Of the 92 HCPs with variable expression, a subset of 24 HCPs exhibitedexpression that correlated with cell age. Most of the proteins withinthis subset are classified as extracellular (71%) or lysosomal (21%) andserve functions related to cell adhesion, proteolysis and metabolism,and angiogenesis. Additionally, a subset of 21 proteins exhibited aproductivity-dependent correlation, with most of these HCPs locatedextracellularly (48%) or in the endoplasmic reticulum (ER, 43%) andserving functions related to protein folding, proteolysis andmetabolism, cell adhesion, and complement and immunity. Nine of the 20productivity-correlated proteins demonstrated maximum expression incells cultured for 366 days, when maximum HCP productivity was observed.One such protein, thrombospondin-1, inhibits cell growth as a tumorsuppressor and therefore may contribute to the reduced viable celldensity in cells cultured for 366 days. The majority (67%) of the nineproteins with expression positively correlated to HCP productivity arelocated in the ER. Increased expression of ER proteins may activateapoptosis and consequently contribute to the observed reduction inviable cell density in cells cultured for 366 days. The increaseddetection of ER proteins in the 366 day cultures likely results fromincreased production and/or secretion of these proteins and notincreased cell lysis because expression of actin, a highly abundantintracellular HCP, did not exhibit statistically significant changesacross the four cell ages (Table 2). This hypothesis is furthersupported by detection of equivalent amounts of L-lactate dehydrogenaseA chain across all four cell ages, as lactate dehydrogenase is anintracellular protein that is commonly measured as a marker of cellhealth.

Of the 92 extracellular CHO HCPs exhibiting variable expression, 34 havepreviously been identified as difficult to remove due to co-elutionand/or product association, and these proteins represent importantcandidates for additional exploration. For example, the majority (63%)of proteins previously identified as mAb-associating also show variableexpression with cell age. Many of these proteins exhibited stronginteractions with at least 3 different mAbs, including clusterin,chondroitin sulfate proteoglycan 4, G-protein coupled receptor 56,neural cell adhesion molecule, nidogen-1, lipoprotein lipase, and SPARC.Furthermore, approximately one-third of proteins previously shown toco-elute during capture by Protein A, mixed-mode, and cationic resinsalso exhibited variable expression with extended culture duration inthis study. For example, 78 kDa glucose-regulated protein has previouslydemonstrated non-specific interactions with Protein A resin resulting incarryover to Protein A eluate, and has been shown to co-elute withproduct fractions during purification by both mixed-mode and cationicresins. In this work 78 kDa glucose regulated protein exhibited variableexpression by both proteomic techniques, including aproductivity-dependent 10-fold expression increase by shotgunproteomics. Additionally, 6 HCPs (alpha-enolase, clusterin, cofilin-1,lysosomal protective protein, peroxiredoxin-1, and procollagen Cendopeptidase enhancer 1) have been identified as purificationchallenges in at least three previous studies in addition todemonstrating variable expression in the present study. This list ofproteins that may challenge purification by other mechanisms in additionto varied expression is not exhaustive because detection of the 92proteins presented in this work may be problematic if the amount ofprotein expressed challenges the limit of detection of proteomic assaysat certain culture durations.

5. Conclusions

Of the hundreds of extracellular CHO HCPs that must be cleared fromtherapeutic products, 118 have previously been reported as difficult toremove because they co-purify during downstream purification. This studyshows that the composition of extracellular HCP impurities changes asCHO cells age, with variably expressed HCPs including a number ofspecies that have previously been identified as difficult to remove. Asbiopharmaceutical manufacturing evolves towards continuousbioprocessing, it is important to consider the impact of extended cellculture on the HCP impurity profile because changes in expression ofdifficult-to-remove impurities may further challenge their clearance indownstream purification. To ensure product quality, purificationoperations must be designed to remove the full range of HCP levelsresulting from such variable expression. Further investigation ofextracellular CHO HCPs with variable expression, particularly those HCPsknown to challenge downstream purification, could improve impurityclearance and enhance the robustness of manufacturing operations.

EXAMPLE 2 Reduction of Lipoprotein Lipase by Small InterferingRibonucleic Acids Limits Degradation of Polysorbate-80

1. Introduction

Polysorbates are a class of non-ionic surfactants that are often addedto biopharmaceutical formulations to improve the stability oftherapeutic proteins by limiting aggregation and surface adsorption. Themajority of monoclonal antibody formulations incorporate polysorbate 80(PS-80), which is composed of polyoxyethylene sorbitan monooleate fattyacid esters, to prolong the shelf-life of drug products. The chemicalstructure of PS-80 is similar to that of a triglyceride, with bothmolecules containing long hydrocarbon chains attached by ester bonds.Degradation of PS-80 by hydrolysis of this ester bond can compromise thestability of therapeutic products.

Certain enzymes, such as lipoprotein lipase (LPL), hydrolyze ester bondswithin triglycerides to form alcohol and fatty acid molecules. Given thestructural similarities between PS-80 and triglycerides, it ishypothesized that LPL may enzymatically degrade PS-80. LPL is a hostcell protein (HCP) that is expressed and secreted by Chinese hamsterovary (CHO) cells. Several factors indicate that LPL may be difficult toremove during biopharmaceutical manufacturing: it exhibits variableexpression with cell age, it product-associates with different mAbs, andit demonstrates similar retention characteristics to mAbs duringpurification with three different polishing resins. Previous work hasshown that recombinant CHO LPL produced in Escherichia coli degradesPS-80 at 37° C.

One method for limiting LPL content in biopharmaceutical drug productsinvolves reducing LPL expression during upstream cell culture bysilencing gene expression using short interfering ribonucleic acid(siRNA) technology. siRNAs are 21-23 nucleotide strands, with a sequencethat is complementary to a specific target messenger RNA (mRNA). ThesesiRNAs are incorporated into the RNA-induced silencing complex (RISC),which binds and cleaves the target mRNA sequence. mRNA cleavage inhibitstranslation and reduces expression of the target protein. The efficiencyof siRNA-mediated gene silencing primarily depends on specific featuresof the siRNA sequence, including G/C content and strand stability. RNAinterference has been demonstrated in CHO cells for bioprocessapplications, such as improving recombinant productivity and extendingculture duration. For example, siRNA-mediated silencing has been appliedto reduce cofilin-1 expression, resulting in increased specificproductivity, and to limit α-1,6 fucosyltransferase expression andconsequently generate defucosylated antibodies with improvedantibody-dependent cellular cytotoxicity.

This research is the first study to apply siRNA technology to reduceexpression of a difficult-to-remove HCP impurity, whose incompleteclearance may result in PS-80 degradation and reduced stability ofbiopharmaceutical products. Here, CHO cells are transfected withLPL-specific siRNAs to limit LPL expression, which is quantified by amultiple selected ion reaction monitoring (MRM) assay. Cell cultureattributes are monitored to explore the impact of reduced LPL expressionon cell growth, and the effect of LPL expression on PS-80 degradation isdemonstrated.

2. Materials and Methods

2.1 CHO Cell Culture

A null CHO-K1 cell line (ATCC, Manassas, Va., USA) was adapted toserum-free, suspension culture in 125 mL shake flasks containing 20-30mL SFM4CHO medium (Hyclone Laboratories Inc., Logan, Utah, USA).Following adaptation, the cells were subjected to extended culture withroutine passaging at 3-5 day intervals in a 37° C. cell cultureincubator with 5% CO2 and 80% relative humidity.

2.2 siRNA Design and Transfection

Adapted CHO cells were exchanged into Opti-MEM medium (LifeTechnologies, Carlsbad, Calif., USA) and independently transfected withthree custom siRNAs (5′-GCAACAATGTGGGCTATGA-3′ (SEQ ID NO: 11),5′-CCTTTCTCCTGATGATGCA-3′ (SEQ ID NO: 13), and 5′-GAAATGATGTGGCCAGGTT-3′(SEQ ID NO: 15)) and a non-specific control (all from Sigma-AldrichChemical Co., St. Louis, Mo., USA). Cells were transfected for 4-6 hoursusing Lipofectamine 2000 (Life Technologies) in 50 mL CultiFlaskbioreactors (Sartorius Stedim Biotech, Göttingen, Germany), andsubsequently diluted in SFM4CHO medium and cultured for 48 hours toenable siRNA-mediated silencing.

Following incubation, cells were counted using a Fuchs Rosenthalhemocytometer with viability determined by a Trypan blue exclusionmethod. The extracellular HCPs were harvested, separated from theresidual cells by centrifugation (180 g, 10 min), analyzed for totalprotein concentration by Bradford assay (Pierce Chemical, Rockford,Ill., USA), and stored at −20° C. until further use.

2.3 Extracellular CHO HCP Preparation

HCPs were precipitated with methanol as described previously (Valente etal. 2014, Biotechnol. J. 9:87-99) and residual detergent was removed byDetergentOUT GBS10-800 detergent removal kit (G-Biosciences, St. Louis,Mo., USA) according to the manufacturer's protocol. Trypsin digestionwas performed as described previously (Valente et al. 2014, Biotechnol.J. 9:87-99). Peptide pellets were resolubilized in 0.1% trifluoroaceticacid (TFA, Fisher Scientific, Fair Lawn, N.J., USA), loaded onto C18ZipTips (EMD Millipore, Billerica, Mass., USA) and eluted in 50%acetonitrile with 0.1% TFA (Fisher Scientific).

2.4 MRM Assay

High pH reversed phase high performance liquid chromatography (RP-HPLC)was performed using an UltiMate 3000 nLC system (Dionex, Sunnyvale,Calif., USA). Digested CHO HCPs were loaded onto a C18 trap column(Dionex) and washed with 150 μL of 2% acetonitrile (MallinckrodtChemicals, Phillipsburg, N.J., USA) in 0.1% formic acid (PierceChemical). Peptides were eluted onto a 0.66 μL C18 column (Dionex) by a26 column linear gradient from 2-49% acetonitrile, followed by anadditional 6 column volumes of 49% acetonitrile. All operations wereperformed at 2.6 min residence time and both mobile phases included 0.1%formic acid. Column eluate was directly injected into a QTrap 4000 (ABSciex, Foster City, Calif., USA) through a nanoSpray II source (ABSciex)with an uncoated fused-silica Pico tip (New Objective, Woburn, Mass.,USA). The instrument was operated in positive ESI ion mode, with sprayvoltage of 2400 V and source temperature of 150° C., with MRM triggeredenhanced resolution scan and enhanced product ion scans. Databasesearches were performed using ProteinPilot software v4.0 (ABSciex)against translations of the CHO genome. Possible MRM transitions weregenerated with Skyline v2.5.0.6157 and monitored through Analyst 1.6.2(AB Sciex), with parameters specified in Table 4. Raw MRM data wereintegrated for peak area and normalized to yeast alcohol dehydrogenase(YAD) transition peak areas. All analysis was performed with threetechnical replicates and two biological replicates.

2.5 PS-80 Degradation Assay

Extracellular CHO HCPs prepared from siRNA transfected cells and controlcultures were independently buffer-exchanged into pH 6.8 with 10 mMCaCl₂ (Sigma-Aldrich Chemical Co). PS-80 (Fisher Scientific) was thenadded to the buffer-exchanged HCPs to a final concentration of 23 mM (3%w/w) and the mixture was incubated at 37° C. for 24 hours with mixing.Enzymatic degradation of PS-80 was measured using the EnzyChrom FreeFatty Acid Kit (Fisher Scientific), which measures the concentration offatty acid released during PS-80 hydrolysis.

3. Results and Discussion

CHO cells were transfected with three different siRNA sequences that arespecific for different regions of the LPL gene as well as a non-specificcontrol siRNA, and results were compared to those for untransfected CHOcells. The extracellular CHO HCPs were analyzed by a MRM assay todetermine the relative amount of LPL in each culture. Transfection withLPL-specific siRNA reduced expression of LPL by 56-72% (FIG. 7A), whilethe total HCP expression remained similar across all five cultures (FIG.7B). This reduction in LPL expression had a limited impact on cellgrowth, with LPL-specific siRNAs demonstrating a 10-19% reduction inaverage cell density (FIG. 8A) and equivalent viability exhibited by allcultures (FIG. 8B). The observed reduction in cell growth (FIG. 8A)following siRNA transfection is in agreement with previous reports usingcofilin-specific siRNA (Hammond and Lee 2011, Biotechnol. Bioeng.109:528-535) and α-1,6 fucosyltransferase-specific siRNA (Mori et al.2004, Biotechnol. Bioeng. 88:901-908); however, transfection with anon-specific control previously demonstrated a reduced growth rate thatwas not observed in this study (Hammond and Lee 2011, Biotechnol.Bioeng. 109:528-535). Transfection with LPL-specific siRNAs reducedexpression of the target HCP while maintaining other attributes that arerelevant to biopharmaceutical manufacturing, demonstrating that thistechnique is suitable for evaluating difficult-to-remove HCPs. Astransfection with siRNAs that are specific for three unrelated proteintargets all resulted in decreased cell growth, this technology is bestsuited for applications that can tolerate a slight decrease in celldensity.

The amount of PS-80 degradation can be determined by monitoring theformation of fatty acids in solution because PS-80 hydrolysis results inthe release of free fatty acids. The extracellular CHO HCP pool fromcontrol cells degraded an average of 0.2% of the initial amount ofPS-80, while the siRNA-transfected cultures with reduced LPL expressionexhibit a level of PS-80 hydrolysis that is statistically insignificant(FIG. 9). Although low, the amount of PS-80 digestion by the controlculture was significantly greater than the assay limit of quantitation(0.1%), suggesting that endogenous CHO LPL is capable of digestingPS-80. This finding is supported by previous reports that PS-80 ishydrolyzed by pancreatin, which contains a mixture of enzymes, includinglipases (Christiansen et al., 2010, Eur. J. Pharm. Sci. 41:376-382).While the exact composition of HCPs from each CHO culture is unknown,the insignificant hydrolysis of PS-80 observed by the HCPs from culturestransfected with LPL-specific siRNA is consistent with LPL functioningas the primary extracellular CHO HCP responsible for PS-80 degradation.

4. Concluding Remarks

LPL is an HCP impurity that is expressed and secreted by CHO cells anddifficult to remove during downstream purification operations because itexhibits product association and similar retention characteristics tomAbs on polishing chromatographic resins. The biological function of LPLis to hydrolyze ester bonds on triglycerides, which are structurallysimilar to PS-80, a surfactant that is added to most biopharmaceuticalformulations to improve stability of the therapeutic product. Thisresearch shows that persistence of LPL through downstream purificationoperations and into the final drug product can degrade PS-80 and thatreducing the expression of LPL in upstream cell culture operations canlimit this degradation. The siRNAs used in this work can be applied tostudy LPL during biopharmaceutical process development or to reduce LPLexpression during therapeutic protein manufacturing. Additionally, thesiRNA-mediated silencing technique shown here is not specific to LPL andcan be applied to study the impact of reduced expression of anydifficult-to-remove HCP impurity.

EXAMPLE 3 PS-80 Digestion by CHO Lipoprotein Lipase from CHO HCP Samples

Samples from CHO HCP were collected from CHO cells, from CHO cellsexpressing each of three different siRNA molecules designed to reduceLPL expression, and from CHO cells expressing a nonspecific siRNAmolecule. The amount of PS-80 degraded was monitored using a fatty acidassay. Pefabloc, an enzyme inhibitor, was added to one sample todetermine whether an inhibitor may block LPL activity and thereforeimpact PS-80 degradation. In FIG. 10A, the amount of PS-80 degraded by asample containing no inhibitor is compared to a sample containingPefabloc inhibitor. The sample without inhibitor demonstrates about 18micromoles of PS-80 degradation compared to the sample with inhibitorwhich demonstrates 8 micromoles of PS-80 degradation. In FIG. 10B, theamount of PS-80 degradation performed by samples derived fromsiRNA-expressing CHO cells is compared to control cells. The controlcells and CHO cells expressing a non-specific siRNA that would not beexpected to significantly reduce PS-80 degradation show about 32 and 42micromoles of PS-80 degraded. Some of the siRNA-expression cells(namely, the siRNA1 and siRNA2 cells) show somewhat less PS-80degradation (28 and 23 micromoles of PS-80 degraded) in this experiment.

EXAMPLE 4 Digestion of Polysorbate by CHO Lipoprotein Lipase Expressedin E. coli

1. Introduction

Polysorbates are nonionic surfactants that are common additives intherapeutic mAb formulations. Of the 30 FDA approved mAbs as of 2012, 19contained polysorbate 80 and 4 contained polysorbate 20. Polysorbatesprotect mAbs from degradation during purification, filtration,freeze-drying, storage and final delivery. They are thought to stabilizehigh-concentration mAb solutions by binding to the product molecules orcompeting with mAbs for surface adsorption. Polysorbate degradation haspreviously been studied and several different routes of polysorbatedegradation in formulations have been identified. Polysorbatedegradation can lead to accelerated product degradation due to increasedaggregation or oxidation due to peroxide formation.

Lipoprotein lipase (LPL) was identified as a difficult-to-remove HCPimpurity in mAb downstream processing. The objectives of the presentwork are to determine if CHO LPL can enzymatically degrade polysorbatesin the pH range of interest for typical mAb formulations (˜pH 5-7).

2. LPL Production

CHO LPL was expressed in E. coli, purified and refolded. Bacterialexpression was used to produce large amounts of LPL rapidly.

2.1 Expression in E. coli

An LPL-containing pET11a plasmid was transformed into BL21-competentcells in SOC broth and plated on ampicillin. Colonies were selected andcultures were grown overnight to seed the production culture.

To confirm LPL protein expression, two cultures were run in autoclaved25 mg/mL LB broth with 100 μg/mL ampicillin at 37° C. The cultures wererun with and without isopropyl β-D-1-thiogalactopyranoside (IPTG)induction. Cells were then pelleted, redissolved in PBS and heated to100° C. after the addition of SDS loading buffer. The material wasloaded and run on a 10% SDS PAGE gel to confirm the expression of CHOLPL. The silver-stained gel is shown in FIG. 11. The culture with IPTGinduction has a band not present in the non-induced culture at slightlygreater than 50 kDa, which is consistent with LPL.

2.2 CHO LPL Purification

After confirmation of LPL expression (FIG. 11), Ni-NTA affinitypurification of LPL was completed. A 750 mL cell culture harvest washomogenized and LPL inclusion bodies were resolubilized in 6 M guanidineHCl. The resulting chromatogram of Ni-NTA affinity purification is shownin FIG. 12. The large A280 signal during loading is due to E. coli HCPimpurities, cell debris and cell culture media additives flowing throughthe column. The step elution using 250 mM imidazole results in a largepeak of eluting LPL with a pronounced tail. Throughout the Ni-NTApurification the mobile phase was maintained at 6 M guanidine HCl.

SDS-PAGE was used to assess the purity of the Ni-NTA LPL elution pool.Samples were buffer-exchanged into PBS prior to loading the gel. Thesilver-stained reducing gel is shown in FIG. 13A. This gel indicates asingle band in both lanes A and B at approximately 50 kDa. The elutionpool (lane B) contains no detectable impurities. The flow-through poolhas a diffuse band at the same molecular weight as the band in theeluate. A western blot was run to confirm the presence of His-tagged LPLand is shown in FIG. 13B. A mouse monoclonal anti-His tag antibody(G020, ABM, Richmond, BC, Canada) was used to detect His-tagged LPL. Theanti-His western confirms that the ˜50 kDa bands in the Ni-NTAflow-through and elution have a His-tag. The presence of LPL in theNi-NTA flow-through is likely due to overloading the column. CHO LPLconcentration was then determined using a Micro BCA™ assay (ThermoScientific, Rockford, Ill.).

After confirming purification, LPL refolding was carried out by rapiddilution with gentle stirring; this method was the most successful ofthose explored and resulted in only limited precipitate formation.

To confirm folding, reverse phase (RP)-HPLC was run with unfolded LPL(LPL solubilized in 6 M guanidine HCl) and refolded LPL. The LPL wasinjected into the C₁₈ column at 1 mL/min with a linear gradient from0-100% acetonitrile in water over 45 minutes. The chromatograms areshown in FIG. 14. The solubilized LPL has two main peaks and manysmaller late-eluting peaks. The refolded LPL also has two main peaks.The majority of refolded LPL is contained in the early-eluting peak,likely due to less solvent exposure of the LPL hydrophobic core. Theunfolded LPL interacts with the C₁₈ column with higher affinity,indicating that it has more hydrophobic character than the folded LPL.Unfortunately there is no CHO LPL standard to confirm correct folding.Without a proper standard this result cannot confirm proper LPL folding,but it does provide insight into the changes in LPL due to the refoldingprocedure. The enzymatic activity that was measured in subsequentsections is further evidence of proper folding of at least asubpopulation of the LPL.

3. Enzymatic Activity of CHO LPL Expressed in E. coli

Measurements of LPL activity against polysorbate 20 and polysorbate 80were carried out in various solution conditions. Refolded LPL wasbuffer-exchanged into the appropriate buffer prior to the activityassay. The conditions investigated were pH 5.0, pH 6.0 and pH 6.8 andthe buffers used were 10 mM sodium acetate, pH 5.0, 10 mM L-histidine,pH 6.0, and 50 mM bis-tris, pH 6.8. Polysorbate 20 or 80 was added tothe buffer-exchanged LPL with a final concentration of 0.23 mM (0.03%).Some samples also had either 10 mM calcium chloride or 10 mM sodiumchloride. The polysorbate and LPL solutions were then incubated at 37°C. with constant mixing for 24 hours.

To assay polysorbate degradation, 270 μM 9-anthryldiazomethane (ADAM) inmethanol was added to each sample in a 3:1 ratio of ADAM solution tosample. The ADAM conjugation was carried out at room temperature usingopaque 1.6 mL Eppendorf tubes with constant mixing for at least 6 hours.Following conjugation the samples were centrifuged at 13,000 g for 6minutes and the supernatant was added to HPLC sample vials. A Viva C18150×4.6 mm column from Restek (Bellefonte, Pa.) was used with a ShimadzuProminence UFLC (Kyoto, Japan). The mobile phase was 97% acetonitrile,3% methanol. Samples were all run in triplicate on the HPLC withinjection volumes of 10 μL and a flow rate of 1 mL/min for 13 minutesper sample. The absorbance at 254 nm was analyzed for thecharacteristics peaks of degraded polysorbate 20, 80 or triglyceride.The EnzyChrom™ Free Fatty Acid Kit was used as a secondary method toconfirm the results of the HPLC assay described by directly measuringthe release of fatty acid by lipase.

4. Results and Discussion

For the ADAM labeling-HPLC assay, a sample chromatogram comparingdegraded and non-degraded polysorbate 80 is shown in FIG. 15. TheADAM-labeled polysorbate degradation product has a characteristic peakat 7 minutes. Activity was measured at pH 5.0, 6.0 and 6.8 in thepresence of either NaCl, CaCl₂ or no additional salt. These conditionswere chosen as they are similar to FDA-approved mAb formulationconditions and Ca²⁺ was previously found to promote the formation ofactive LPL dimers.

The experimentally measured degradation rates of polysorbate 80 areshown in FIG. 16. Overall, there is measurable polysorbate 80degradation in almost all of the conditions tested. Although thesedegradation rates were measured at an unrealistically high temperaturefor mAb storage, it is helpful to put the measured rates into context.For example, in a formulation containing 10 ppm LPL, a degradation rateof 0.1 μM polysorbate/μM LPL/hr translates to a polysorbate degradationcorresponding to concentrations of approximately 0.02-0.03% per year, soannual degradation amounts are comparable to the total polysorbatecontent in formulated samples. Degradation rates were found to increasewith increasing pH, consistent with prior work on lipase catalysis thatshowed maximum rates at higher pH. The addition of the two salts has aminimal effect, contrary to previous findings. The most extensivedegradation was found at pH 6.8 with 10 mM CaCl₂, but similar rates werefound with NaCl and no additional salt, so neither salt appearsnecessary for active LPL against polysorbate 80. The degradation ratesmeasured here were similar to previous findings with pancreatin.

LPL degradation rates of polysorbate 20 are shown in FIG. 17 for thesame conditions as for polysorbate 80. Overall, LPL activity is muchlower using polysorbate 20, which is less frequently added toformulations, but still commonly used. In contrast to the polysorbate 80degradation results, most of the conditions with measurable degradationwere at pH 5.0; polysorbate 20 at pH 6.8 with 10 mM CaCl₂ was the onlyother condition where degradation was detected. Polysorbate 20degradation rates at pH 5.0 increase significantly upon the addition ofeither NaCl or CaCl₂, consistent with observations in previous work.

These findings demonstrate the possibility of polysorbate degradationdue to CHO LPL presence in final formulations. HCPs with enzymaticactivity have previously been observed to result in mAb degradation. Themeasured degradation rates show relative trends among differentconditions, but the implications of the nominal rates in a bioprocessingenvironment cannot readily be interpreted meaningfully due to a numberof significant differences. In particular, the E. coli-produced LPLlacks glycosylation and is probably not completely folded. Also, thesestudies were completed at an elevated temperature. Rates of LPLdegradation of polysorbate at typical storage temperatures were notmeasured.

5. Conclusions

The polysorbate degradation studies reported here confirm that CHO LPLrecombinantly produced in E. coli or natively produced by CHO cells candegrade either polysorbate 20 or 80 by ester hydrolysis in mAbformulation conditions. The optimal solution conditions for degradationof polysorbate 80 were consistent with previous findings for lipases. Itwas also found that for polysorbate 20, LPL had higher activity at pH5.0 with either NaCl or CaCl₂ present at 10 mM.

These results demonstrate both the difficulty of removing LPL during mAbpurification processes as well as the danger of not removing LPL.Degradation of polysorbate in formulations is a previously identifiedproblem that should be avoided at all costs. At the very least LPLshould be monitored through downstream purification and in finalformulations; tracking LPL is not overly difficult and could provideinsight into otherwise unexplained product degradation.

EXAMPLE 5 Genome Editing of Host Cells

There are several approaches available to knock out the presence ofspecific target genes in a genome. So-called genome editing tools employthe CRISPR-Cas9 mechanism of gene editing, the use of TALENs or the useof zinc finger nucleases. We are studying the use of the CRISPR-Cas9 andalso TALEN based approaches to knock out the presence of lipoproteinlipase from the genome of CHO cells. Because lipoprotein lipase is notbelieved to be an essential gene or protein, the knock out of this geneshould allow cells to remain viable. Moreover, it will preventexpression of this particular host cell protein which is known to bedifficult to remove. By eliminating expression of this gene, the proteincannot be expressed and it cannot coelute or copurify with recombinantproteins of interest. Because it will not be present in the purifiedrecombinant protein, it will not be able to degraded polysorbates suchas PS-80.

1. Transfection of hCas9+sgRNA and LPL-TALEN Plasmids in SuspensionSerum-Free CHO Cells

For CRISPI-Cas9 knock out, four different sgRNA target molecules arebeing tested for their ability to target and eliminate LPL from the CHOgenome while not having any significant off target effects. In addition,a non-specific sgRNA molecule is also being tested as a control.

CRISPR Transfection: 1×10⁶ Suspension serum-free CHO cells weretransfected with 1 μg hCas9 and 1 μg sgRNA plasmids using Lipofectamine2000, according to manufacturer's protocol. Cells were incubated at 37°C. for 3 days.

Experiment Plasmids 1 2 μg Cas9 only 2 Cas9, sgRNA_1 3 Cas9, sgRNA_2 4Cas9, sgRNA_3 5 Cas9, sgRNA_4 6 Cas9, sgRNA_non-specific

TALEN Transfection: 1×10⁶ suspension serum-free CHO cells weretransfected with 1 μg LPL-TAL left and 1 μg LPL-TAL right plasmids usingLipofectamine 2000, according to manufacturer's protocol. Cells wereincubated at 37° C. for 3 days.

2. Selection of Cells by Exposure to Selection Reagents

3 days after transfection, 0.5×10⁶ CRISPR-transfected cells were exposedto 600 μg/mL Geneticin and 600 μg/mL Zeocin for 2 days. Genomicextraction was performed with the remaining cells using Qiagen's DNeasyBlood & Tissue Kit.

Cells were counted 2 days after exposure to selection reagents. However,0% viability were seen in all CRISPR-transfected cells. Cells may bemore vulnerable to the toxicity of the selection reagent aftertransfection. To determine the optimal concentration of selectionreagents, tests with concentrations of selection reagents aftertransfection is needed.

3. Serial Dilution and Semi-Solid Media Plating of Transfected Cells

5 days after transfection, TALEN-transfected cells were serially dilutedon a 96-well plate at a density of 0.5 cell/well. Cells were also platedin triplicates at 400 cells/mL in semi-solid media, supplemented with 4mM L-glutamine, on 6-well plates. These plates will incubate at 37° C.for 10 days.

TABLE 1 LPL siRNA sequences Sense/ Target Antisense siRNA Design StartSequence Sense GCAACAAUGUGGGCU  926 GCAACAATGTGGGCT AUGAdTdT ATGA(SEQ ID NO: 1) (SEQ ID NO: 11) Antisense UCAUAGCCCACAUUG  926TCATAGCCCACATTG UUGCdTdT TTGC (SEQ ID NO: 2) (SEQ ID NO: 12) SenseCCUUUCUCCUGAUGA  588 CCTTTCTCCTGATGA UGCAdTdT TGCA (SEQ ID NO: 3)(SEQ ID NO: 13) Antisense UGCAUCAUCAGGAGA  588 TGCATCATCAGGAGA AAGGdTdTAAGG (SEQ ID NO: 4) (SEQ ID NO: 14) Sense GAAAUGAUGUGGCCA  392GAAATGATGTGGCCA GGUUdTdT GGTT (SEQ ID NO: 5) (SEQ ID NO: 15) AntisenseAACCUGGCCACAUCA  392 AACCTGGCCACATCA UUUCdTdT TTTC (SEQ ID NO: 6)(SEQ ID NO: 16) Sense CUUUGUCAUCGAGAA 1272 CTTTGTCATCGAGAA GAUUdTdT GATT(SEQ ID NO: 7) (SEQ ID NO: 17) Antisense AAUCUUCUCGAUGAC 1272AATCTTCTCGATGAC AAAGdTdT AAAG (SEQ ID NO: 8) (SEQ ID NO: 18) SenseGAAGUAUUGGGAUCC  653 GAAGTATTGGGATCC AGAAdTdT AGAA (SEQ ID NO: 9)(SEQ ID NO: 19) Antisense UUCUGGAUCCCAAUA  653 TTCTGGATCCCAATA CUUCdTdTCTTC (SEQ ID NO: 10) (SEQ ID NO: 20)

TABLE 2 Proteins with variable expression, which were identified by MSfrom 2DE images. Statistical significance determined by ANOVA ofrelative protein spot volume from three biological replicates ofproduction culture sourced from a single cryopreserved stock for eachcell age. Protein identifications from translations of the CHO genomeunless otherwise noted. Spot # Accession # Protein Name p-value  1gi|344252604 Laminin subunit gamma-1 0.277  2 gi|344244798 Nidogen-10.032  3 gi|304510 78 kDa glucose-regulated protein 0.013  4gi|344242104 Sulfated glycoprotein 1 0.145  5 gi|344246008 Lysyloxidase-like 1 0.018  6 gi|16508150 ERP57 protein 0.013  7 gi|344250216Procollagen C-endopeptidase 0.016 enhancer 1  8 gi|145567052 Serineprotease 0.060  9 gi|115497814 Nucleobindin-1 0.212 10 gi|344241583Lysosomal protective protein 0.004 11 gi|344259113 Pigmentepithelium-derived factor 0.126 12 gi|344251524 Nucleobindin-2 0.008 13gi|344248735 Cathepsin D 0.003 14 gi|761724 Beta-actin 0.429 15gi|344254255 Cathepsin B 0.188 16 gi|344240379 Vesicularintegral-membrane 0.950 protein VIP36 17 gi|3442536563-phosphoinositide-dependent 0.922 protein kinase 1 18 gi|344249681Clusterin 0.706 19 gi|344242456 Complement C1r-A subcomponent 0.053 20gi|344242993 Collagen alpha-1(III) chain 0.095 21 gi|344242455Calcium-dependent serine 0.177 proteinase 22 gi|344258664Metalloproteinase inhibitor 1 0.153 23 gi|344255270 V-type proton ATPasesubunit S1 0.102 24 gi|899229b Thrombospondin-1 0.002 25 gi|7434045Glutathione transferase class pi 0.023 26 gi|81917543 Peroxiredoxin-10.078 27 gi|62948096 Basement membrane-specific 0.002 heparan sulfateproteoglycan core protein 28 gi|1344237299 Immunoglobulin superfamily0.352 member 8 29 gi|344238428 Peptidyl-prolyl cis-trans 0.294 isomeraseC 30 gi|344256956 Cofilin-1 0.045 31 gi|344252163 Nucleoside diphosphatekinase B 0.028 32 gi|344252164 Nucleoside diphosphate kinase A 0.683

TABLE 3 CHO HCPs previously identified as purification challenges byother mechanisms in addition to demonstrating varied expression withprolonged cultivation duration in this study. Product Protein Mixed-Cation or Protein Name Assoc. A mode MMC 78 kDa glucose-regulatedprotein x x x Acid ceramidase x Alpha-enolase x x x x Basementmembrane-specific x heparan sulfate proteoglycan core protein Beta2-microglobulin x x Cathepsin D x x Cathepsin Z x x Chondroitin sulfateproteoglycan 4 x Clusterin x x x Cofilin-1 x x x Collagen alpha-1(III)chain x Complement C1r-A subcomponent x Galectin-3-binding protein xGlutathione transferase class pi x x G-protein coupled receptor 56 xHeat shock protein HSP 90-beta x Insulin-like growth factor- x bindingprotein 4 Laminin subunit alpha-5 x Laminin subunit beta-1 x x Lamininsubunit gamma-1 x Legumain x Lipoprotein lipase x x Lysosomalalpha-glucosidase x Lysosomal protective protein x x x Metalloproteinaseinhibitor 1 x x N(4)-(beta-N-acetylglucosaminyl)- x L-asparaginaseNeural cell adhesion molecule 1 x Nidogen-1 x x Peptidyl-prolylcis-trans x isomerase B Peroxiredoxin-1 x x x ProcollagenC-endopeptidase x x x enhancer 1 Putative phospholipase B-like 2 xSerine protease x x SPARC x

TABLE 4 MRM assay parameters. Scan Target peptide SEQ Precursor ProductProduct time CE sequence ID (m/z) (m/z) ion (ms) (V) LPLITGLDPAGPNFEYAEAPSR 21 1002.987   793.424  y72⁺ 20 72.9ITGLDPAGPNFEYAEAPSR 21 1002.987   430.271  y42⁺ 20 67.9ITGLDPAGPNFEYAEAPSR 21 1002.987   359.204  y32⁺ 20 52.9 EPDSNVIVVDWLYR22  852.933  1162.662  y92⁺ 20 44.4 EPDSNVIVVDWLYR 22  852.933 1063.594  y82⁺ 20 44.4 EPDSNVIVVDWLYR 22  852.933   950.509  y72⁺ 2044.4 ITGLDPAGPNFEYAEAPSR 21  668.994   793.424  y73⁺ 20 37.8ITGLDPAGPNFEYAEAPSR 21  668.994   630.331  y63⁺ 20 37.8ITGLDPAGPNFEYAEAPSR 21  668.994   430.241  y43⁺ 20 37.8LSPDDADFVDVLHTFTR 23  974.476   661.352  y52⁺ 20 56.3 GLGDVDQLVK 24 522.29    873.478  y82⁺ 20 30.5 GLGDVDQLVK 24  522.29    701.429  y62⁺20 30.5 GLGDVDQLVK 24  522.29    602.361  y52⁺ 20 30.5 YAD ANELLINVK 25 507.3031  699.4763 y62⁺ 20 32.9 ANELLINVK 25  507.3031  586.3923 y52⁺20 32.9 ANGTTVLVGMPAGAK 26  693.8741  730.3916 y82⁺ 20 42.8ANGTTVLVGMPAGAK 26  693.8741  631.3232 y72⁺ 20 42.8 EALDFFAR 27 484.7454  655.3198 y52⁺ 20 31.7 EALDFFAR 27  484.7454  540.2929 y42⁺ 2031.7 VVGLSTLPEIYEK 28  483.2729  778.3981 y63⁺ 20 32   VVGLSTLPEIYEK 28 483.2729  552.3028 y43⁺ 20 32  

TABLE 5 Candidate protein A wash solutions adapted from previous work(Shukla and Hinckley, 2008). Wash number pH Wash contents 1 4.4 50 mMcitrate, 1% polysorbate 80 2 4.4 50 mM citrate, 1M urea 3 9.0 25 mMtris, 10% isopropyl alcohol, 3M urea 4 9.0 25 mM tris, 1% polysorbate80, 10% isopropyl alcohol, 3M urea

What is claimed:
 1. A non-naturally occurring cultured host cellcomprising a nucleic acid sequence encoding a recombinant protein,wherein the host cell expresses the recombinant protein and is amammalian cell selected from the group consisting of CHO, 3T3, BHK,HeLa, NS0, and HepG2, and wherein at least one copy of an endogenousgene encoding an endogenous lipoprotein lipase is knocked out from thegenome of the host cell.
 2. The non-naturally occurring cultured hostcell of claim 1, wherein the host cell is CHO.